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Temporo-spatial distribution and transcriptional profile of retinal microglia in the oxygen-induced retinopathy mouse model

ABSTRACT: Myeloid cells such as resident retinal microglia or infiltrating blood-derived macrophages accumulate in areas of retinal ischemia and neovascularization (RNV) and modulate neovascular eye disease. Their temporo-spatial distribution and biological function in this process, however, remain unclarified. We determined the extent of microglia proliferation and macrophage infiltration in areas of ischemia and RNV using Cx3cr1CreERT2:Rosa26-tdTomato mice and assessed the transcriptional profile of microglia in the oxygen-induced retinopathy (OIR) mouse model. We show that microglia are the predominant myeloid cell population in areas of RNV. Thirty percent of retinal microglia were EdU-positive indicating considerable expansion of local microglia. RNA-Seq revealed an enrichment of processes related to cell division and chemokine receptor binding. We propose that activated retinal MG alter their transcriptional profile, exhibit proliferative ability and are by far the most frequent myeloid cell population in areas of RNV in the OIR model thus presenting a potential target for future therapeutic approaches.

ORGANISM(S): Mus musculus

PROVIDER: GSE132731 | GEO | 2020/03/22

REPOSITORIES: GEO

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Project description:Background: Retinal neovascularization (RNV) as a result of retinal ischemia, such as in proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP), can lead to vitreous hemorrhage, tractional retinal detachment, and irreversible loss of vision if left untreated. Although panretinal laser photocoagulation and anti-vascular endothelial growth factor (VEGF) injections are efficient treatment modalities, a significant number of patients do not respond to either treatment. This clinical finding suggests that other, previously unrecognized mediators and signaling pathways may contribute to RNV development and could represent valuable targets for future treatments. Methods: In search of phylogenetically conserved angiogenic mediators, we examined the transcriptional profile of murine RNV from C57BL/6J mice (n=14) in the oxygen-induced retinopathy (OIR) model as well as human RNV membranes from PDR patients (n=7), who had undergone vitrectomy and compared them with corresponding control tissues (n=13, 10 respectively). Genes that were differentially expressed (DEGs) between RNV and control samples were identified for human and murine samples and their associated gene ontology (GO) clusters analyzed. Lastly, human and murine DEGs were compared to identify phylogenetically conserved factors. Findings: Transcriptional profiles of murine RNV showed that DEGs linked to the activation of the innate immune system (Msn, Cd34), extracellular matrix organisation (Col4a1, Gfap), and regulation of angiogenesis (Col4a2, Fgf2) were significantly upregulated both at the ischemic, preproliferative stage of the disease (OIR p14) as well as at the proliferative stage (OIR p17). While similar GO terms were upregulated in human RNV, only a small overlap in DEGs between both species was detected. Phylogenetically conserved mediators upregulated in both murine and human RNV included ANGPT2, S100A8, MCAM, EDNRA, MRC1, and CCR7. Interpretation: This study identifies phylogenetically conserved inflammatory and pro-angiogenic mediators that are significantly upregulated in both murine and human RNV. Among them, MCAM, ENDRA and MRC1 emerged as the most upregulated, phylogenetically conserved DEGs not yet implicated in human RNV, thus representing potential new treatment targets for ischemic retinal diseases.

Project description:Background: Proliferative diabetic retinopathy (PDR) is hallmarked by the formation of retinal neovascularization (RNV) membranes, which can lead to a tractional retinal detachment, the primary reason for severe vision loss in end-stage disease. The aim of this study was to characterize the molecular and cellular features of RNV in order to unravel potential novel drug treatments for PDR. Methods: A total of 42 patients undergoing vitrectomy for PDR, macular pucker or macular hole (control patients) were included in this study. The surgically removed RNV and epiretinal membranes were analyzed by RNA sequencing, single-cell based Imaging Mass Cytometry and conventional immunohistochemistry. Since macrophages were found to be abundant in RNV tissue, vitreal macrophages, also known as hyalocytes, were isolated from the vitreous of patients with PDR by flow cytometry, cultivated and characterized by immunhistochemistry. A bioinformatical drug repurposing approach was applied, in order to identify novel drug options for end-stage diabetic retinopathy disease. Results: The in-depth transcriptional and single-cell protein analysis of diabetic RNV tissue samples revealed an accumulation of endothelial cells, macrophages and myofibroblasts as well as an abundance of secreted ECM proteins such as SPARC, FN1 and several types of collagen in RNV tissue. The immunohistochemical staining of cultivated vitreal hyalocytes from patients with PDR showed that hyalocytes express α-SMA (alpha-smooth muscle actin), a classic myofibroblast marker. According to our drug repurposing analysis, imatinib emerged as a potential drug option for future treatment of PDR. Conclusion: This study delivers the first in-depth transcriptional and single-cell proteomic characterization of RNV tissue samples. Our data suggest an important role of hyalocyte-to-myofibroblast transdifferentiation in the pathogenesis of diabetic vitreoretinal disease and suggests their modulation as a novel possible clinical approach.

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Temporo-spatial distribution and transcriptional profile of retinal microglia in the oxygen-induced retinopathy mouse model

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